The first molecular characterization of the Tunisian-Algerian endemic Trifolium julianiBatt. and Trifolium squarrosum L. from Dyr El Kef, North West Tunisia

In Tunisia, clovers represent a valuable genetic resource due to their economic and ecological importance in forage and pastoral systems, particularly within meslin cropping systems. However, certain species, such as Trifolium squarrosum , are increasingly threatened with extinction owing to the lack of conservation and management strategies. This study proposes a clarification of the distinction between Trifolium squarrosum and Trifolium juliani . To substantiate this differentiation, molecular characterization was performed using two types of molecular markers: ISSR (Inter Simple Sequence Repeats) and SRAP (Sequence-Related Amplified Polymorphism). A total of 10 ISSR primers generated 70 polymorphic bands across the two Trifolium species, demonstrating a high polymorphism percentage (PPB = 80.46%), with notable variation in band patterns among primers. Conversely, 10 SRAP primer combinations yielded 46 polymorphic bands (PBP = 73.02%). While both marker systems effectively revealed molecular polymorphism, ISSR markers exhibited slightly higher efficiency. Analysis of molecular variance (AMOVA) indicated that genetic diversity primarily partitioned between the two species, reflecting low gene flow and high genetic differentiation. Principal component analysis (PCA) and hierarchical clustering which further supported that distinction, revealed clear genetic structure among the clover species. This structure was also corroborated by robust clustering and high assignment probabilities in the Bayesian inference analysis. This study underscores the efficacy of ISSR and SRAP markers in assessing genetic diversity and differentiating clover species, providing valuable insights into their genetic relationships.